Amyotrophic lateral sclerosis (ALS) is a poorly understood neurodegenerative disease with no effective treatment. ALS is connected with military service and veterans are at a higher risk of contracting this debilitating disease. Mutations in several genes have been discovered to cause a subset of familial ALS, including copper- zinc superoxide dismutase (SOD1), fused in sarcoma (FUS), and TAR DNA-binding protein 43 (TDP-43). Studying familial ALS using the well-defined genetic models will provide critical insights into the disease pathology as well as novel targets for future therapeutic development. This project is focused on the RNA binding protein FUS that has been implicated in both familial and sporadic ALS. Since FUS mutations were found in a subset of familial ALS patients in 2009, mislocalization of mutant FUS in the cytoplasm has been considered a hallmark of this subset (Type 6) of ALS. However, protein inclusions containing wild-type FUS have been reported in multiple forms of familial and sporadic ALS, suggesting that FUS plays an important role in a larger portion of ALS beyond the Type 6 familial ALS. Protein inclusions can be caused by protein over-production, inefficient protein degradation, protein mislocalization, or a combination of these defects. However, the regulation of FUS protein expression and degradation remains poorly understood. We have published that it is critical to maintain proper levels of FUS inside the nucleus and that either elevated or reduced levels of FUS causes similar degenerative phenotypes in Drosophila models. Thus we propose that maintaining appropriate FUS protein homeostasis is critical to attenuate FUS toxicity and motor neuron dysfunction. Since little is known about how FUS gene transcription and protein turnover are regulated, discoveries in this project will fill this knowledge gap and advance the field significantly. The specific hypotheses to be tested are: FUS gene transcription is regulated by the Jak/Stat signaling cascade (Aim 1) while FUS degradation is largely mediated by the ubiquitin-proteasome system (Aim 2). Moreover, we will test the hypothesis that FUS ubiquitination regulates its RNA binding and subcellular localization (Aim 3). We have carried out preliminary studies to show that the transcription of FUS is indeed controlled by Jak/Stat signaling and that Jak/Stat signaling attenuates FUS toxicity in Drosophila models. We also show that the degradation of FUS is mediated by the ubiquitin-proteasome system and that FUS ubiquitination is regulated by a specific E3 ligase named FBXW7. Moreover, a dominant negative mutant FBXW7 decreased FUS ubiquitination, delayed FUS degradation and changed FUS subcellular localization. Therefore each of these three specific hypotheses is supported by our preliminary results. These inter-related pathways play a critical role in regulating FUS function and alterations in any of them will likely contribute to the pathogenesis of ALS. The results from this study will help understand how to target FUS in motor neurons, providing a new avenue to attenuate FUS toxicity in future therapeutic developments. The proposal is constructed based on a number of novel observations, which leads to a novel hypothesis that that FUS protein biosynthesis and turnover can be modulated to attenuate FUS toxicity in ALS. An array of techniques will be employed to test the hypothesis in a combination of in vitro and in vivo models. The results from the proposed studies should yield potential new targets that can be translated into novel treatments for veteran and other ALS patients.